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The analysis of circulating tumor cells using lab-on-chip technologies could determine whether cancer is about to spread or monitor the progress of treatment. The objective of CTC analysis is to detect, identify and count these cells in peripheral blood. By characterization of the trapped CTC their genetic and pathophysiological relevance to the primary tumor can be identified. The project aims at creating a microfluidic chip for isolating circulating tumor cells based on nano-magnetic technology.

The microfluidic chip will be tunable using nano-magnetic structures such as ferrofluids, self-organized magnetic particles, or magnetically active polymeric actuators. The key and unique feature of the proposed microfluidic chip will be that the device can be tuned to maximize the sensitivity and yield for a specific type of cancer. Tuning is done by a magnetic field created outside the micro-fluidic channel. The key advantage of the proposed technology is that only a single device is needed to investigate various types of cancer.

The tunable CTC chip can be applied for cancer biology research and clinical cancer management including including the detection, diagnosis and monitoring of cancer.

1.2 Project duration

Start: Q4, 2010

End: Q3, 2012

1.3 Summary WP1

Workpackage 1: Ferrofluid based structures

Ferromagnetic fluids for building an adaptable periodic structure should be used. Ferrofluids consist of spherical ferromagnetic nanoparticles suspended typically in water building a colloid. In an external magnetic field, the ferrofluid is deflected toward the gradient of the magnetic flux density and building 3 dimensional structures.

By applying a periodical arrangement of permanent magnets or electrical conducting wires, the magnetic fluid settles down at the points of the highest magnetic flux density building a periodic structure. By modulation of an external electrical field or by adjustment of the electrical current through the wires, the size and shape of the structure (magnetic fluid) can be modified. For CTC enrichment the surface of the ferromagnetic particles should be functionalized with EpCAM antibodies to enable a CTC capturing onto the surfaces of the ferrofluidic 3D structure.

After removing the magnets or switching off the electrical current, the ferrofluidic 3D structures collapse and can be washed out together with the trapped cells to a cell counting chamber. After threefold labeling (DAPI, cytokeratin, APC) for differentiation of captured CTC and leucocytes, CTC counting can be done by fluorescence microscopic imaging.

Metastasis is the late and dangerous state of any cancer types. One of the reasons for this state is the colonization of CTCs at distant organs, which were released from a primary tumor into the bloodstream. The finding of CTCs in blood has been documented in literature for a while, more than a century ago by T.R. Ashworth, an Austrian pathologist who first reported this type of cells [1]. Meanwhile, there are various methods describing CTC detection. However, due to its rare presence in blood compared to other blood cells, different assays are used to detect CTCs. There is still need for improvement and development, especially in the enrichment steps, to increase the isolation success rate. The detection and analysis of CTCs may play important roles in diagnosis and treatment of cancer patients.

Different enrichment approaches were listed in categories: size-based (filtration, density gradient) and immunomagnetic enrichment. A summary of those methods were done by Marianna Alunni-Fabbroni et al [2] is as following:

Figure 1: Summary of different CTC enrichment approaches.

The PhD thesis of Kristian Smistrup [3] was very impressive, he did a very comprehensive study about using magnetic field strength as the main separating tool in microfluidic chips.

Recently, there was a published article from Antone-Emmanuel Saliba et al [4]. These articles together with the CTC-chip [5] confirm the right direction for this project from the beginning, that using a permanent magnet outside a microfluidic chip to trap magnetic beads covered with appropriate antibodies would work with high efficiency for CTCs enrichment, detection and analysis.

2.2 Microfluidic chip design:

Middle November 2010, there was a meeting with Prof. Velekoop and his group at the Institute for Sensors and Actuator Systems - Technical University Vienna (TU Vienna) as his group will be contributing on this project with the strong understanding and long experience in microfluidic chip designing and processing.

The chip consists of several components: a housing; microstructures of walls, seeding point carriers or seeding point structures; tubing and pump system for in-out fluid.

(For the first models, this third component will not be discussed yet for simplicity)

A first model of the housing for the microfluidic chip was already proposed by the group at TU Vienna. This housing was used for some existing projects there.

For building the microstructures, SU-8 technique was suggested. SU-8 is a commonly used epoxy-based photoresist [6, 7]. It is a very viscous polymer that can be spun or spread over a thickness ranging from 0.1 micrometer up to 2 millimeters and still be processed with standard contact lithography. It can be used to pattern high aspect ratio (&gt;20) structures [5] due to the fact that it is highly transparent in the ultraviolet range. When SU-8 is exposed to UV light its molecular chains are cross-linked, causing the SU-8 to solidify. SU-8 was originally developed as a photoresist for microelectronics industry, to provide a high-resolution mask for fabrication of semiconductor devices. It is now mainly used in the fabrication of microfluidics (mainly via soft lithography, but also with other imprinting techniques such as nanoimprint lithography) and microelectromechanical systems parts. As its known biocompatible properties [5,8], SU-8 is often used in bio- microelectromechanical systems (MEMS).

A mixture of SU8 photoresist with ferromagnetic micro/nano particles is magnetically active and can be used as the structural material for components of MEMS [9]. This composite ferromagnetic photoresist (FPR) offers three advantages for fabricating magnetically-responsive microstructures [10].

* The structure can be made at once. Mold fabricating, electroplating or subsequent lithography and etching steps are not necessary.

* While films made by sputtering or dry etching of polymer/magnetic material mixtures, the thickness of the structure can be adjusted over a wide range: from submicron to hundreds of microns.

* FPR can used to fabricate flexible ferromagnetic microstructures and mechanisms. Young s modulus of the SU8 (3 109 Nm?2) is significantly smaller than that of other magnetic materials used for micromachining, such as nickel (207 109Nm?2) or iron (208 109 N m?2). Using the weighted average, it was estimated that the Young s modulus of the FPR prepared by suspending nickel nanoparticles (5% by weight) in photosensitive epoxy SU8 (95%) to be 13 109 N m?2. The small Young s modulus of FPR means that a suspended structure made of this material is compliant, and has a larger range of motion than one made from nickel or iron under the same actuation force.

Our chip microstructures will be in the range of 0-250 m and therefore the processing steps can follow the above table to choose the specific SU-8 photoresist and the according spin speed for wanted microstructure thicknesses.

Study from Nicolae Damean et al. [10] also showed that, since the wavelength of light used for exposure (365 or 405 nm) was larger than the size of the suspended nanospheres (80 150 nm) in the photoresist, diffraction and scattering of light allowed for exposure of the volume of photoresist immediately behind the nanospheres. Multiple scattering of light from individual nanospheres, and reflections from aggregates, helped further penetration of the light into the FPR. We will suggest this exposure range to TU Vienna for the building process of our first microfluidic chips.

2.3

Hands-on with raw materials and models

In the normal lab environment, we proceeded some experimental studies to get the feelings with magnetic particles (nano and micrometer size), to mix our own solution for magnetic particles to prevent its aggregation in normal condition, and to build our models with the uncomplicated materials and set up.

2.3.1 Experiments with Ferrofluids

A commercially available ferrofluid (ferrofluidic kit 003665, Ferrotec, USA) has been used. It is a suspension of magnetic nanoparticles (spherical, diameter 10nm) in a carrier liquid. The particles are covered by a detergent, which avoids agglomeration of the particles.

We wanted to mix the fluid with a translucent liquid, to get a fluid which can be pumped through a tube and through our device for enrichment of the magnetic particles at points of high magnetic flux density (seeding points).

We found, that this ferrofluid is not suitable for our experiments, because the ferrofluid is attracted by plastics and our tubes are made of plastic. The wall of the tubes would be coated with the fluid and we would have the particles distributed all over the tube.

Therefore we had to make our own ferrofluid with nanoparticles.

2.3.2 Experiments with nanoparticles

We used commercially available nanoparticels (diameter 50nm covered with PVP as surfactant, Sigma Aldrich). These nanoparticles have to be mixed with distilled water (pH 7). Ultrasound has been used to disperse the particles in the solution. Because of agglomeration we added a surfactant.

We found albumin to be the best surfactant. It is both, hydrophobic and hydrophilic, which makes it the ideal surfactant for our nanoparticle solution. Another advantage of albumin is, that there are no contraindications to use it with human blood. This fluid was used in the following experiments.

2.3.3 Applying a magnetic field:

Two different methods have been tested:

* Small permanent magnets:

We have used small magnets with the diameter of 2mm and height 4mm and positioned under our fluid with nanoparticles (described above). We found an enrichment of magnetic particles around the magnets.

* Seeding points in combination with a high magnetic field of a permanent magnet:

The seeding points are made by nanoparticle based structures, which are placed in small holes of a polycarbonate slide.

Dimension of the first prototype:

Depth of the holes: 250 m

Diameter of the holes: 600 m

Distance between centers of the holes: 5mm

The nanoparticles have been placed in the holes and covered by a transparent tape. The fluid with nanoparticles (described above) was added on top of the seeding points. Underneath the seeding points we placed a permanent magnet (different strength from 20 to 100mT). No enrichment of magnetic particles could be observed.

The far distance between the seeding points and their small dimensions were probably the reason for these results.

First trials have been made to simulate our experiment with COMSOL in 2D.

Following is the result of our first trial in COMSOL, we can see distribution of iron oxide particle in the fluid which concentrates more on the seeding points. The magnetic force acting to the ferromagnetic particles is defined by

where m is the magnetic moment of the particles and B is the magnetic flux density. Therefore as higher the gradient of the magnetic flux density as higher is the magnetic force acting to the magnetic particles. In our setup the highest magnetic filed gradient is found at the margin of the seeding points where the simulation shows the highest particle agglomeration.

Figure 5: Distribution of iron oxide particles simulated by COMSOL

3 Conclusions

Studies from literature till now show that this project is going on the right tract and has promising outcomes. The studies gave us also some hints to realize the beginning ideas, for instant to localize and stabilize bead structures by building seeding points. Comparisons between theoretical simulation in COMSOL and lab experiments will be made when there are enough experiments and simulation results. The collaboration with TU Vienna will help to speed up experiments in real models of chips in the near future.

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